1. Field of the Invention
The present invention relates to a method of reducing the severity of host vs graft reaction (HVGR) by down-regulating autoimmunity to heat shock protein hsp60. In particular, the present invention relates to the use of hsp60 protein or hsp60 epitope peptides to suppress graft rejection by down-regulating hsp60 autoimmunity.
2. Description of the Background Art
Autoimmune disorders, e.g., insulin-dependent diabetes mellitus (IDDM or type I diabetes), multiple sclerosis, rheumatoid arthritis and thyroiditis, are characterized by reactivity of the immune system to an endogenous antigen, with consequent injury to tissues. These immune responses to self-antigens are maintained by the persistent activation of self-reactive T lymphocytes.
The 60 kDa heat shock protein (hsp60) is a stress protein expressible in all of the cells of the body. Nevertheless, healthy individuals manifest a high frequency of autoimmune T cells specific for hsp60 self-epitopes. Normal healthy mice and human beings have been shown to have T cells targeted at their self hsp60 antigen (Kaufmann, 1990; Kaufmann et al., 1994; Young, 1989; Cohen, 1992b). However, these autoimmune T cells are also involved in T cell mediated autoimmune diseases: a high concentration of T cells targeted at self hsp60 antigen have been found in the autoimmune lesions of human chronic arthritis (Cohen, 1991; Res et al., 1989; van Eden et al., 1989), multiple sclerosis (Selmaj et al., 1991), experimental autoimmune encephalomyelitis (Selmaj et al., 1991) and adjuvant arthritis (Hogervorst et al., 1992). Anti-hsp60 T-cells have also been shown to play a role in diabetes mellitus in the non-obese diabetic (NOD) mouse model (Elias et al., 1990; Elias et al., 1991; Elias et al., 1994; Elias et al., 1995; Birk et al., 1996a; Birk et al., 1993; Cohen , 1991).
NOD mice spontaneously develop type I diabetes caused by autoimmune T cells that attack the insulin-producing .beta. cells of the islets. The autoimmune attack is associated with T-cell reactivity to a variety of self-antigens including a peptide of the 60 kDa heat shock protein (hsp60) and peptides of glutamic acid decarboxylase (GAD). Thus, for example, spontaneous diabetes developing in the NOD/Lt strain of mice could b e treated with hsp60 (U.S. Pat. No. 5,114,844) or with antigenic fragments thereof, such as the peptide designated p277 corresponding to positions 437-460 of the human hsp60 sequence (PCT Patent Publication No. W090/10449); variants of the p277 peptide in which one or both cysteine residues at positions 6 and/or 11 have been replaced by valine and/or the Thr residue at position 16 is replaced by Lys (see PCT Publication WO96/19236); or various other peptides such as those designated p12 and p32, corresponding to positions 166-185 and 466-485, respectively, of the human hsp60 sequence (see PCT application PCT/US96/11375). Note that the hsp60 protein was formerly designated hsp65 but is now designated hsp60 in view of more accurate molecular weight information; by either designation the proteins are the same.
Immunization to hsp60 or peptide p277 in an appropriate adjuvant is known to induce IDDM (WO90/10449) when the immunization triggers a TH1 response. However, vaccination with hsp60 or peptide p277 without an effective adjuvant, and preferably with a tolerogenic carrier or, more preferably, with a TH2-inducing active carrier, can produce a resistance to the autoimmune process of IDDM. Subcutaneous administration of p277 in incomplete Freund adjuvant (IFA; mineral oil) led to the arrest of disease progression in young NOD mice (Elias et al., 1991) or in 12-17 week old NOD mice with advanced insulitis (Elias et al., 1994 and 1995). Both the human (Elias et al., 1994 and 1995) and mouse (Birk et al., 1996a) variants of p277 were effective. NOD mice transgenic for the mouse hsp60 gene on an MHC class II promoter showed down-regulation of their spontaneous T-cell proliferative response to p277 and a significant proportion of the mice were spared the development of diabetes (Birk et al., 1996b). Moreover, administration of p277 to C57BL/KsJ mice aborted the development of autoimmune diabetes in mice that had earlier received a very low dose of the .beta.-cell toxin streptozotocin (STZ); treatment of these mice with a peptide of the GAD65 molecule was not effective (Elias et al., 1996). Preferred TH2-inducing active carriers for use in the administration of such hsp60 fragments are certain fat emulsions, such as Intralipid or Lipofundin (see PCT/US96/11373).
Furthermore, T cells reactive to hsp60 and other hsp molecules have been isolated from cardiac allografts (Moliterno et al., 1995) as well as from other inflammatory lesions (Selmaj et al., 1991; Mor et al., 1992). Thus, anti-hsp60 autoimmune T cells accumulate at sites of inflammation. Donor-specific alloreactive T lymphocytes exhibiting such characteristics as cytolytic activity and lymphokine production are believed to mediate transplant rejection where hsp-reactive T cells may play a role in the immune cascade of the inflammatory process in transplant rejection (Moliterno et al., 1995).
It has recently been discovered by the laboratory of the present inventors that the successful treatment of the autoimmune process in IDDM by administration of the peptide p277 in oil is caused by the effect of this treatment in aborting TH1-type autoimmunity to several different antigens and instead activating p277 autoimmunity into a TH2 mode. T cells of the CD4 "helper" type have been divided into two groups, TH1 and TH2, by the characteristic cytokines they secrete when activated (Mosmann et al, 1989). TH1 cells secrete IL-2, which induces T cell proliferation, and cytokines such as IFN-.gamma., which mediate tissue inflammation. TH2 cells, in contrast, secrete IL-4 and IL-10. IL-4 helps cells secrete antibodies of certain IgG isotypes and suppresses the production of TH1 inflammatory cytokines (Abas et al., 1994). IL-10 indirectly inhibits TH1 activation by affecting antigen-presentation and inflammatory cytokine production by macrophages (Moore et al., 1993). It is the TH1 cells which contribute to the pathogenesis of organ-specific autoimmune diseases. TH1-type responses also appear to be involved in other T cell mediated diseases or conditions, such as contact dermatitis (Romagnani, 1994). Thus, a disease with a spectrum of autoreactivities can be turned off with a single peptide capable of inducing a T cell cytokine shift.
Transplantation is presently the treatment of choice for organ failure. During the 1980s, the discovery of cyclosporin A played a role in the significant increase in graft survival. But the use of transplants is still limited because of the acute immune rejection phenomenon (host vs graft rejection, or HVGR) which may destroy the transplanted tissue within days to months after the surgery. Control and regulation of HVGR are crucial to the development of better transplantation methods.
Nonspecific immunosuppressive therapy in an adult patient is usually through cyclosporin, started intravenously at the time of transplantation, and given orally once feeding is tolerated. Typically, methylprednisone is started also at the time of transplantation, then reduced to a maintenance dose. Azathioprine is also often used in conjunction with prednisone to achieve adequate immunosuppression. Whereas the objective of immunosuppression is to protect the transplant, general or excessive immunosuppression may lead to undesirable complications, e.g., opportunistic infections and potential malignancies. The toxicity of the immunosuppressive drugs is dose-dependent.
Means to diminish or eliminate HVGR have been long sought in the art. Any process capable of reducing the vigor of the rejection process and reducing the doses of immunosuppressive drugs needed to maintain the graft would be a significant advance in this art.
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